Protein Dynamics Affect O ₂ -Stability of Group B [FeFe]-Hydrogenase from <i>Thermosediminibacter oceani</i>

In the pursuit of sustainable "green" energy generation, [FeFe]-hydrogenases have attracted significant attention due to their ability to catalyze hydrogen production. However, the sensitivity of these enzymes to O₂ is a major obstacle for their application as biocatalysts in energy conversion technologies. In the search for an O₂-stable [FeFe]-hydrogenase, we identified the hydrogenase ToHydA from <i>Thermosediminibacter oceani</i> that belongs to the rarely characterized Group B (M2a) [FeFe]-hydrogenases. Our findings demonstrate that ToHydA exhibits remarkable O₂-stability, even under prolonged O₂ exposure. By characterizing site-directed mutagenesis variants, we found that the highly conserved proton-transporting active site cysteine residue protects the H-cluster from O₂-induced degradation by forming the H_inact state. The additional cysteine residue in the TSCCCP motif of ToHydA, a feature unique to Group B (M2a) [FeFe]-hydrogenases, enhances the flexibility of that motif and facilitates the formation of the H_inact state. Moreover, ToHydA possesses unique features, including the formation of an unusual H_inact resting state that distinguishes the enzyme from other [FeFe]-hydrogenases. Our atomistic molecular dynamics simulations reveal a previously unrecognized cluster of hydrophobic residues centered around the proton-transporting cysteine-bearing loop. This structural feature appears to be a common molecular characteristic in hydrogenases that form the O₂-protected H_inact state. By exploiting these molecular features of ToHydA, future research can aim to rationally design hydrogenases that combine high catalytic activity with enhanced O₂-stability to develop more efficient and durable catalysts.